Developmental Patterns of Seizure Susceptibility in Inbred Strains of Mice BETTY S. DECKARD B. LIEFF Department of PsychoEogy
K. SCHLESINGER J. C. DeFRIES Department of Psychology arid Institute for Behavioral Genetics University o f Colorado Boulder, CoIorado
Samples of mice from cach of 6 inbred strains were tested for audiogenic and electroconvulsive seizures at 5 ages (14, 21, 28, 35, and 42 days). A moderately largc within-strain correlation (.67) was found, indicating that developmental patterns of susceptibility to audiogenic and electroconvulsive seizures are similar within each strain. The finding of an even larger between-strain correlation (.91) indicated tkat strains which are highly susceptible t o audiogenic scizures are also likely to bc highly susceptible t o electroconvulsive seizures. In a 2nd experiment, whole brain norepinephrine and serotonin were assayed in each of 5 inbred strains at 21 and 42 days of age. Results were consistent with the hypothesis that levels of these amines are inversely related t o seizure susccptibility. Mice from strains which were susceptible to seizures at 21 days of age had significantly lower levels of norepinephrine and serotonin in brain than did 42-day-old, seizureresistant animals.
Susceptibility to audiogenic (sound-induced) seizures depends upon a number of variables including the genotype of the organism being tested. The importance of genetic factors has been demonstrated by strain comparisons (e.g., Coleman & Schlesinger, 1965; Fuller & Sjursen, 1967), by selective breeding experiments (e.g., Frings & Frings, 1953; Nellhaus, 1963), and by single-gene analyses (Collins & Fuller, 1968; Ginsburg, Cowen, Maxson, & Sze, 1967). An important question related to the generality of this phenomenon is whether animals which are genetically predisposed to audiogenic seizures are also more susceptible to seizures produced b y other means, especially to drug- and electrically-induced seizures. Positive correlations between susceptibility to audiogenic seizures and t o seizures produced electrically and by various drugs (pentylenetetrazol, nicotine, caffeine, strychnine, physostigmine, thiosemicarbazide, and picrotoxin) have been reported. These data are mainly derived from experiments performed on DBA/2J and C57BL/6J mice (summarized in reviews by Fuller & Wimer (1 9661 and Schlesinger & Received for publication 9 December 1974 Rcvised for publication 19 February 1975 Developmental Psychobiology. 9( I ) : 17-24 (1 976) 0 1 9 7 6 by John Wiley & Sons, Inc.
17
18
DECKARD ET AL.
Uphouse [1972]). An exception t o this finding has been reported by Swinyard, Castellion, Fink, and Goodman (1963), who failed to observe a cori-elation between susceptibility t o audiogenic and pentylenetetrazol-induced seizures in O’Grady mice. The primary objective of the present study was to investigate possible covariation o f susceptibility to audiogenic and electroconvulsive seizures as a function of age in mice of 6 inbred strains. Although genetically detertnined susceptibility t o audiogenic seizures has becn studied extensively, the mechanisms which mediate this phenotype remain unclear. Coleman (1960) was the first t o propose the hypothesis that differences in levels of the biogenic amines and gamma-aminnbutyric acid (GABA) might be causally related l o susceptibility to audiogenic seizures. He also hypothesized that these differences in biogenic amines between dilute and liondilute strains of mice were attritutable to differences in phenyalanine metabolism caused by different genes at the (1 locus in Linkage Group 11. Howevcr, the d locus is not necessarily or causally related t o seizure risk [Schlesinger, Elston, & Boggan, 19661 .) Coleman’s ( I 960) first hypothesis is supported by the following observations: ( 1 ) Seizure-susceptible DBA/2J mice have lower levels o f 5-hydroxytryptamine (serotonin, 5-IfT) xnd norepinephrine (NE) in the brain than do seizure-resistant C57BlJ6.J mice. but only at 21 days of age, which corresponds t o the period of maximal seizure risk in DBA!2J animals (Schlesinger, Boggan, & Freedman, 1965). Kellogg (1971) has confirmed this finding with respect to levels of 5-HT in mice of these strains; in addition, she reported differences in 5-HT turnover in these animals. (2) Pharmacological treatments which raise the levels of the biogenic amines in these mice protect agaiiist audiogenic seizures, whereas treatments wluch Iower levcls of these compounds increase seizure risk. Similar data have been obtained with respect t o levels o f CABA (Lehrnan, 1970; Schlesinger, Boggan, & Freedman, 1968, 1970). (3) Diets deficient in pyridoxine (Vitamin B6), w h c h lower levels of GABA in brain tissue, increase susceptibility t o audiogenic and electroconvulsive seizures, whereas supplemental pyridoxine protects against these seizures (Schlesinger & Schreiber, 1969; Schlesinger & Uphouse, 1973). (4) Levels of 5-HT in the central nervous system (CNS) and susceptibility to audiogenic seizures exhibit a diurnal fluctuation, and the 2 rhythms are correlated (Schreibcr & Schlesinger, 1971). The secondary objective of the present study was to test further thc adequacy of this hypothesis by comparing levels of 5-HT and NE in young (i.e., scizure-susceptible) and old (i.e., seizure-resistant) animals in each of 5 strains of mice.
Subjects A total of 145 DBA/lJ, 120 DBA/2J, 160 C57BL/bJ, 110 C57BL/lOJ, 114 BDP/J, and 100 P/J n i c e (Mus rnusculi*.s) were used as experimental subjects. T h e origin and degree of inbreeding of these animals have been described previously (Jay, 1963). The animals were bred in our laboratory from stock obtained from The Jackson l;lboratory, Bar Harbor, Maine. Until used in the study, all mice were maintained under standard laboratory conditions of temperature (24 2°C) and controlled lighting ( I 2-hr light: 13-hr dark cycle; lights on at 0700 hours) with ad lib access to Purina Mouse Breeder _+
DEVELOPMENT OF SEIZURE SUSCEPTIBILITY
19
Chow and tap water. In view of the circadian alterations in seizure susceptibility, all tests were performed between the 4th and 8th hour of the daily light cycle (1 100 and 1500 hours, respectively). Each mouse was tested only once, and approximately equal numbers of male and female animals were used in each experiment. The exact number of mice used in each experiment, as well as the ages of the animals at time of testing, are given below.
Audiogenic Seizure Tests Animals were tested for susceptibility t o audiogenic seizures as follows: Subjects of the appropriate age were placed, 1 at a time, into a large chromatography jar (45.5 cm high, 29.5 cm in diameter) and given 30 sec to adapt. A 12.5-cm electric bell, generating 116 dB of noise at the level of the mouse, was then sounded for 60 sec or until the animal stopped breathing. During this interval the animal was observed and records made of the incidence of wild running and of clonic, tonic, and lethal seizures. For purposes of statistical analysis all mice were given a “seizure severity score”, computed as follows: no response = 0 ; wild running = 1 ; clonic seizure = 2 ; tonic seizure = 3; and lethal seizure = 4.
Electroconvulsive Seizure Tests Each mouse was placed into a plastic restraining device, and ear clips were carefully attached. It was stimulated with an 833.3-cps square wave of .2 sec duration, having a negative and positive pulse width of .3 msec and a delay of .6 msec between each cycle (total period = 1.2 msec). The current intensity used in all seizure tests was 5.0 mA. Incidences of clonic, tonic, and lethal seizures were recorded, and each mouse was gven a seizure xeverity score based on its response. Responses were scored as follows: no response = 0; clonic seizure = 2 ; tonic seizure = 3; and lethal seizure = 4.
Norepinephrine and Serotonin Determinations For biochemical analyses the animals were sacrificed by decapitation, the brains were quickly removed, and 2 tissue samples were taken. Cortical tissue was obtained by making cuts originating between the olfactory lobes and continuing dorsally to the corpus callosum. Other cortical tissue, including the insula, was then pared away and included with the cortical sample. Subcortical tissue comprised all the remainder of the brain. After dissection, the parts of brain were weighted to the nearest .01 g. The 5-HT and NE determinations were made using the spectrophotofluorometric technique described by Maickel, COX,Saillant, and Miller (1968). All reagents used in these assays were purchased from Regis Chemical Company.
Results The developmental profiles of susceptibility to both aubogenic and electroconvulsive seizures are given in Figure 1. In the majority of the strains tested, a specific developmental pattern was associated with susceptibility t o audiogenic seizures. In the DBA/IJ, DBA/2J, and C57BL/10J strains, peak susceptibility occurred at either 21 or 28
20
DECKARD ET AL. - 7 T - ’
C 5 7 ELI6 J
C 57BLIlO J
A I , ,, I 14 21 2 8 3 5 4 2
,
-
i
14 21
2 8 3 5 42
A G E ( I N DAYS)
Fig. 1. Developmental profiles of susccptibility to audiogenic (filled circles) and electroconvulsive (open circles) seizures in 6 inbred mouse strains.
days of age. In animals of the BDP/J strain, relatively high seizure-severity iscores were obtained at 21, 28, and 35 days of age. Mice o f the P/J strain wei-e relatively seizure susceptible at all ages, whereas those of the C57BL/6J strain were seizure resistant. The severity scores, the number of :iiiirnals in m c h experiment, and the details of the seizure tests in terms of the percentage of animals of each strain and age exhibiting the various types of seizure responses are summarized in Table 1. With respect to susceptibility to electroconvulsive seizures, a strain-specific developmental pattern again appeared (see Fig. 1 & Table 1). In all of the s t r i n s tested, the animals were maximally susceptible to electroconvulsive seizures at one particular age, except for DBA/lJ and DBA/2J mice which were susceptible at both 21 and 28 days. Given the stimulus parameters used in these tests, 14-day old mice were resistant to these seizures. Inspection of the data presented in Figure 1 suggests that susceptibilities t o audiogenic and electroconvulsive seizures are correlated responses in mice o f these 6 inbred strains. The withn-strain correlation between these 2 measures was .67 (df= 1 X, p < .(It), indicating that responses on these measures covary as a function of age. In other words, developmental patterns of susceptibility t o audiogenic and electroconvulsive seizures are much the same within a particular mouse strain. The data also :;uggest that strains which are susceptible t o one type of seizure are also susceptible to other modes of seizure induction. The overall between-strain correlation was .91 (df = 4, p < .05). This correlation indicates that if animals of a particular strain are h g h l y susceptible to audiogenic seizures, they will also be highly susceptible to electroconvulsive seizures. The data obtained on levels of 5-HT and NE in brains of these animals are summarized in Table 2. Animals from strains w h c h are susceptible t o seizures at 21 days of age have generally lower levels of 5-HT and NE in the brain at 21 days than at 42 days of age when they are seizure resistant. The DBAlIJ, DBA/2J, and C57BL/10J mice “ere found to have significantly lower brain levels of both 5-HT and NE at 21 days o f age. The
DEVELOPMENT OF SEIZURE SUSCEPTIBILITY
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21
22
DECKARD ET AL. TABLE 2. Brain Levels of NE and 5-HT in Mice o f 5 Inbred Strains. ~
Strm
~
Region
21 Days
~
USA/t.l
C57UL/l 0J
I{DP/.l
P
42 Days
~
NE/Cortex NE/Subcortex 5-H T/Cort ex 5-HT/Subcortex
218 5 385 t 354 i 486 ?
18.03 20.25 17.72 18.35
336 5 441 5 480 t 629 t
22.32 19.62 10.12 9.17
NB/Cortcx NE/Subcortex 5-II'I'/Cortex 5-l-fT/Subcortex
217 317 347 375
t
21.20 17.72 25.95 25.95
31 2 t 443 t 528 t 528 i
36.7 I 19.62 19.94 15.51
NE/Cortex NE/SUbcortcx 5-HT/Cortex 5-HT/Subcor t cx
228 t 402 5 363 t 472 t
12.66 25.63 19.30 30.38
316 c 35.12 4 4 8 i 23.42 373 t 21.20 492 i 14.87
'C .01 ns ns ns
NE/Cortex NE/Subcortex 5-HT/Cortex 5-1ITISuhcortex
208 t 11.39 374 i 33.23 298 t 26.90 458 rt 22.15
319 i 17.40 470 t 13.92 480 t 18.99 593 5 17.40
:.
N E/Cort ex NE/Subcortcx S-tlT/Cortex 5-HT/Subcortex
242 t 29.43 314 c 19.94 421 i 26.58 459 rt 19.62
379 rt 525 5 448 t 477 t
t f
t
14.50 43.50 53.00 50.00
K. SCHLESINGER J. C. DeFRIES Department of Psychology arid Institute for Behavioral Genetics University o f Colorado Boulder, CoIorado
Samples of mice from cach of 6 inbred strains were tested for audiogenic and electroconvulsive seizures at 5 ages (14, 21, 28, 35, and 42 days). A moderately largc within-strain correlation (.67) was found, indicating that developmental patterns of susceptibility to audiogenic and electroconvulsive seizures are similar within each strain. The finding of an even larger between-strain correlation (.91) indicated tkat strains which are highly susceptible t o audiogenic scizures are also likely to bc highly susceptible t o electroconvulsive seizures. In a 2nd experiment, whole brain norepinephrine and serotonin were assayed in each of 5 inbred strains at 21 and 42 days of age. Results were consistent with the hypothesis that levels of these amines are inversely related t o seizure susccptibility. Mice from strains which were susceptible to seizures at 21 days of age had significantly lower levels of norepinephrine and serotonin in brain than did 42-day-old, seizureresistant animals.
Susceptibility to audiogenic (sound-induced) seizures depends upon a number of variables including the genotype of the organism being tested. The importance of genetic factors has been demonstrated by strain comparisons (e.g., Coleman & Schlesinger, 1965; Fuller & Sjursen, 1967), by selective breeding experiments (e.g., Frings & Frings, 1953; Nellhaus, 1963), and by single-gene analyses (Collins & Fuller, 1968; Ginsburg, Cowen, Maxson, & Sze, 1967). An important question related to the generality of this phenomenon is whether animals which are genetically predisposed to audiogenic seizures are also more susceptible to seizures produced b y other means, especially to drug- and electrically-induced seizures. Positive correlations between susceptibility to audiogenic seizures and t o seizures produced electrically and by various drugs (pentylenetetrazol, nicotine, caffeine, strychnine, physostigmine, thiosemicarbazide, and picrotoxin) have been reported. These data are mainly derived from experiments performed on DBA/2J and C57BL/6J mice (summarized in reviews by Fuller & Wimer (1 9661 and Schlesinger & Received for publication 9 December 1974 Rcvised for publication 19 February 1975 Developmental Psychobiology. 9( I ) : 17-24 (1 976) 0 1 9 7 6 by John Wiley & Sons, Inc.
17
18
DECKARD ET AL.
Uphouse [1972]). An exception t o this finding has been reported by Swinyard, Castellion, Fink, and Goodman (1963), who failed to observe a cori-elation between susceptibility t o audiogenic and pentylenetetrazol-induced seizures in O’Grady mice. The primary objective of the present study was to investigate possible covariation o f susceptibility to audiogenic and electroconvulsive seizures as a function of age in mice of 6 inbred strains. Although genetically detertnined susceptibility t o audiogenic seizures has becn studied extensively, the mechanisms which mediate this phenotype remain unclear. Coleman (1960) was the first t o propose the hypothesis that differences in levels of the biogenic amines and gamma-aminnbutyric acid (GABA) might be causally related l o susceptibility to audiogenic seizures. He also hypothesized that these differences in biogenic amines between dilute and liondilute strains of mice were attritutable to differences in phenyalanine metabolism caused by different genes at the (1 locus in Linkage Group 11. Howevcr, the d locus is not necessarily or causally related t o seizure risk [Schlesinger, Elston, & Boggan, 19661 .) Coleman’s ( I 960) first hypothesis is supported by the following observations: ( 1 ) Seizure-susceptible DBA/2J mice have lower levels o f 5-hydroxytryptamine (serotonin, 5-IfT) xnd norepinephrine (NE) in the brain than do seizure-resistant C57BlJ6.J mice. but only at 21 days of age, which corresponds t o the period of maximal seizure risk in DBA!2J animals (Schlesinger, Boggan, & Freedman, 1965). Kellogg (1971) has confirmed this finding with respect to levels of 5-HT in mice of these strains; in addition, she reported differences in 5-HT turnover in these animals. (2) Pharmacological treatments which raise the levels of the biogenic amines in these mice protect agaiiist audiogenic seizures, whereas treatments wluch Iower levcls of these compounds increase seizure risk. Similar data have been obtained with respect t o levels o f CABA (Lehrnan, 1970; Schlesinger, Boggan, & Freedman, 1968, 1970). (3) Diets deficient in pyridoxine (Vitamin B6), w h c h lower levels of GABA in brain tissue, increase susceptibility t o audiogenic and electroconvulsive seizures, whereas supplemental pyridoxine protects against these seizures (Schlesinger & Schreiber, 1969; Schlesinger & Uphouse, 1973). (4) Levels of 5-HT in the central nervous system (CNS) and susceptibility to audiogenic seizures exhibit a diurnal fluctuation, and the 2 rhythms are correlated (Schreibcr & Schlesinger, 1971). The secondary objective of the present study was to test further thc adequacy of this hypothesis by comparing levels of 5-HT and NE in young (i.e., scizure-susceptible) and old (i.e., seizure-resistant) animals in each of 5 strains of mice.
Subjects A total of 145 DBA/lJ, 120 DBA/2J, 160 C57BL/bJ, 110 C57BL/lOJ, 114 BDP/J, and 100 P/J n i c e (Mus rnusculi*.s) were used as experimental subjects. T h e origin and degree of inbreeding of these animals have been described previously (Jay, 1963). The animals were bred in our laboratory from stock obtained from The Jackson l;lboratory, Bar Harbor, Maine. Until used in the study, all mice were maintained under standard laboratory conditions of temperature (24 2°C) and controlled lighting ( I 2-hr light: 13-hr dark cycle; lights on at 0700 hours) with ad lib access to Purina Mouse Breeder _+
DEVELOPMENT OF SEIZURE SUSCEPTIBILITY
19
Chow and tap water. In view of the circadian alterations in seizure susceptibility, all tests were performed between the 4th and 8th hour of the daily light cycle (1 100 and 1500 hours, respectively). Each mouse was tested only once, and approximately equal numbers of male and female animals were used in each experiment. The exact number of mice used in each experiment, as well as the ages of the animals at time of testing, are given below.
Audiogenic Seizure Tests Animals were tested for susceptibility t o audiogenic seizures as follows: Subjects of the appropriate age were placed, 1 at a time, into a large chromatography jar (45.5 cm high, 29.5 cm in diameter) and given 30 sec to adapt. A 12.5-cm electric bell, generating 116 dB of noise at the level of the mouse, was then sounded for 60 sec or until the animal stopped breathing. During this interval the animal was observed and records made of the incidence of wild running and of clonic, tonic, and lethal seizures. For purposes of statistical analysis all mice were given a “seizure severity score”, computed as follows: no response = 0 ; wild running = 1 ; clonic seizure = 2 ; tonic seizure = 3; and lethal seizure = 4.
Electroconvulsive Seizure Tests Each mouse was placed into a plastic restraining device, and ear clips were carefully attached. It was stimulated with an 833.3-cps square wave of .2 sec duration, having a negative and positive pulse width of .3 msec and a delay of .6 msec between each cycle (total period = 1.2 msec). The current intensity used in all seizure tests was 5.0 mA. Incidences of clonic, tonic, and lethal seizures were recorded, and each mouse was gven a seizure xeverity score based on its response. Responses were scored as follows: no response = 0; clonic seizure = 2 ; tonic seizure = 3; and lethal seizure = 4.
Norepinephrine and Serotonin Determinations For biochemical analyses the animals were sacrificed by decapitation, the brains were quickly removed, and 2 tissue samples were taken. Cortical tissue was obtained by making cuts originating between the olfactory lobes and continuing dorsally to the corpus callosum. Other cortical tissue, including the insula, was then pared away and included with the cortical sample. Subcortical tissue comprised all the remainder of the brain. After dissection, the parts of brain were weighted to the nearest .01 g. The 5-HT and NE determinations were made using the spectrophotofluorometric technique described by Maickel, COX,Saillant, and Miller (1968). All reagents used in these assays were purchased from Regis Chemical Company.
Results The developmental profiles of susceptibility to both aubogenic and electroconvulsive seizures are given in Figure 1. In the majority of the strains tested, a specific developmental pattern was associated with susceptibility t o audiogenic seizures. In the DBA/IJ, DBA/2J, and C57BL/10J strains, peak susceptibility occurred at either 21 or 28
20
DECKARD ET AL. - 7 T - ’
C 5 7 ELI6 J
C 57BLIlO J
A I , ,, I 14 21 2 8 3 5 4 2
,
-
i
14 21
2 8 3 5 42
A G E ( I N DAYS)
Fig. 1. Developmental profiles of susccptibility to audiogenic (filled circles) and electroconvulsive (open circles) seizures in 6 inbred mouse strains.
days of age. In animals of the BDP/J strain, relatively high seizure-severity iscores were obtained at 21, 28, and 35 days of age. Mice o f the P/J strain wei-e relatively seizure susceptible at all ages, whereas those of the C57BL/6J strain were seizure resistant. The severity scores, the number of :iiiirnals in m c h experiment, and the details of the seizure tests in terms of the percentage of animals of each strain and age exhibiting the various types of seizure responses are summarized in Table 1. With respect to susceptibility to electroconvulsive seizures, a strain-specific developmental pattern again appeared (see Fig. 1 & Table 1). In all of the s t r i n s tested, the animals were maximally susceptible to electroconvulsive seizures at one particular age, except for DBA/lJ and DBA/2J mice which were susceptible at both 21 and 28 days. Given the stimulus parameters used in these tests, 14-day old mice were resistant to these seizures. Inspection of the data presented in Figure 1 suggests that susceptibilities t o audiogenic and electroconvulsive seizures are correlated responses in mice o f these 6 inbred strains. The withn-strain correlation between these 2 measures was .67 (df= 1 X, p < .(It), indicating that responses on these measures covary as a function of age. In other words, developmental patterns of susceptibility t o audiogenic and electroconvulsive seizures are much the same within a particular mouse strain. The data also :;uggest that strains which are susceptible t o one type of seizure are also susceptible to other modes of seizure induction. The overall between-strain correlation was .91 (df = 4, p < .05). This correlation indicates that if animals of a particular strain are h g h l y susceptible to audiogenic seizures, they will also be highly susceptible to electroconvulsive seizures. The data obtained on levels of 5-HT and NE in brains of these animals are summarized in Table 2. Animals from strains w h c h are susceptible t o seizures at 21 days of age have generally lower levels of 5-HT and NE in the brain at 21 days than at 42 days of age when they are seizure resistant. The DBAlIJ, DBA/2J, and C57BL/10J mice “ere found to have significantly lower brain levels of both 5-HT and NE at 21 days o f age. The
DEVELOPMENT OF SEIZURE SUSCEPTIBILITY
=030c
' O d O -*ci
99990 3 0 0
P I N N
939QC
0 0 0 0 0
0000
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0 0 0 0 C r - * C I
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0 0 0 0
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30 3c W N -
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oaoo P - r - W N
03.9 N N
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99999
C-33EC
01
u d
-
* - 1 O v , P 1 N
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042
21
22
DECKARD ET AL. TABLE 2. Brain Levels of NE and 5-HT in Mice o f 5 Inbred Strains. ~
Strm
~
Region
21 Days
~
USA/t.l
C57UL/l 0J
I{DP/.l
P
42 Days
~
NE/Cortex NE/Subcortex 5-H T/Cort ex 5-HT/Subcortex
218 5 385 t 354 i 486 ?
18.03 20.25 17.72 18.35
336 5 441 5 480 t 629 t
22.32 19.62 10.12 9.17
NB/Cortcx NE/Subcortex 5-II'I'/Cortex 5-l-fT/Subcortex
217 317 347 375
t
21.20 17.72 25.95 25.95
31 2 t 443 t 528 t 528 i
36.7 I 19.62 19.94 15.51
NE/Cortex NE/SUbcortcx 5-HT/Cortex 5-HT/Subcor t cx
228 t 402 5 363 t 472 t
12.66 25.63 19.30 30.38
316 c 35.12 4 4 8 i 23.42 373 t 21.20 492 i 14.87
'C .01 ns ns ns
NE/Cortex NE/Subcortex 5-HT/Cortex 5-1ITISuhcortex
208 t 11.39 374 i 33.23 298 t 26.90 458 rt 22.15
319 i 17.40 470 t 13.92 480 t 18.99 593 5 17.40
:.
N E/Cort ex NE/Subcortcx S-tlT/Cortex 5-HT/Subcortex
242 t 29.43 314 c 19.94 421 i 26.58 459 rt 19.62
379 rt 525 5 448 t 477 t
t f
t
14.50 43.50 53.00 50.00
